A long-pursued goal of structural biology has been to determine the structure of membrane proteins in their membrane environment, ideally in their various functional states as defined for example by ion gradients and membrane potentials. In the proposed work we will apply a new, general method for single-particle cryo-EM structure determination of membrane proteins reconstituted into liposomes. The method is called random spherically-constrained (RSC) single-particle reconstruction and involves the acquisition and processing of images of lipid vesicles containing the protein of interest and having the desired transmembrane gradients imposed. The aims of the project are first to optimize the specimen, imaging and processing strategies for this process, and then apply it to structure determination of Kv and BK potassium channels in their various states. The goal is to observe the conformational changes that underlie the channel gating processes, in particular the rearrangements of voltage-sensor domains of both channel types and the calcium-sensing gating ring of the BK channel.